LOCUS       sequence-367948-        4598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template to generate via PCR two gRNAs for expression in S. 
            cerevisiae. The PCR product from pEasyG2_hph recombines in vivo with
            a PCR product from pEasyG2_mic..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4598)
  AUTHORS   Jacobus AP, Barreto JA, de Bem LS, Menegon YA, Fier I, Bueno JGR, 
            Dos Santos LV, Gross J
  TITLE     EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9
            Applications in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2022 Nov 18;11(11):3886-3891. doi: 
            10.1021/acssynbio.2c00348. Epub 2022 Oct 18.
  PUBMED    36257021
REFERENCE   2  (bases 1 to 4598)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.2c00348"; journalName: "ACS Synth Biol"; date: 
            "2022-11-18- 18"; volume: "11"; issue: "11"; pages: "3886-3891"
COMMENT     Sequence Label: pEasyG2_hph
FEATURES             Location/Qualifiers
     source          1..4598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     847..866
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        847..865
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(919..952)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(1015..2590)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1015..1212)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1218..2246)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTSPRA
                     KE"
     promoter        complement(2247..2590)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(2622..2641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2665..2933
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      3020..3106
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3198..3225
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(3303..3350)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(3363..4157)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3474..3493)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4084..4103
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        4471..4545
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4550..4569
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccagtg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttaa cctggcttat
      841 cgaaattaat acgactcact atagggagac cggcagatcc gcggccgcat aggccactag
      901 tggatctgat atcacctaat aacttcgtat agcatacatt atacgaagtt atattaaggg
      961 ttctcgagag ctcgttttcg acactggatg gcggcgttag tatcgaatcg acagcagtat
     1021 agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc
     1081 atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa
     1141 tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt
     1201 tttattgtca gtactgatta ttcctttgcc ctcggactag tgctggggcg tcggtttcca
     1261 ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt
     1321 tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc
     1381 caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg
     1441 cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag
     1501 tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc
     1561 tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca
     1621 ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg
     1681 cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg
     1741 tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca
     1801 cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg
     1861 ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc
     1921 agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag
     1981 gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat
     2041 gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc
     2101 aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag
     2161 agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc
     2221 gcggtgagtt caggcttttt acccatggtt gtttatgttc ggatgtgatg tgagaactgt
     2281 atcctagcaa gattttaaaa ggaagtatat gaaagaagaa cctcagtggc aaatcctaac
     2341 cttttatatt tctctacagg ggcgcggcgt ggggacaatt caacgcgtct gtgaggggag
     2401 cgtttccctg ctcgcaggtc tgcagcgagg agccgtaatt tttgcttcgc gccgtgcggc
     2461 catcaaaatg tatggatgca aatgattata catggggatg tatgggctaa atgtacgggc
     2521 gacagtcaca tcatgcccct gagctgcgca cgtcaagact gtcaaggagg gtattctggg
     2581 cctccatgtc gctgattacc ctgttatccc tacactctca gtacaatctg ctctgatgcc
     2641 gcatagttaa gccagccggg taattctttg aaaagataat gtatgattat gctttcactc
     2701 atatttatac agaaacttga tgttttcttt cgagtatata caaggtgatt acatgtacgt
     2761 ttgaagtaca actctagatt ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt
     2821 tttcacaccc tacaatgttc tgttcaaaag attttggtca aacgctgtag aagtgaaagt
     2881 tggtgcgcat gtttcggcgt tcgaaacttc tccgcagtga aagataaatg atcatttccc
     2941 cgaaaagtgc cacctgcatc gatggccccc gatggtagtg tggggtctcc ccatgcgaga
     3001 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     3061 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     3121 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     3181 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ggccagtgcc
     3241 aagcttgcat gcagattgca gcattacacg tcttgagcga ttgtgtaggc tggagctgct
     3301 tcgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc aagatcccct
     3361 tattagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
     3421 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
     3481 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
     3541 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
     3601 acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
     3661 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
     3721 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
     3781 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
     3841 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
     3901 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
     3961 cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
     4021 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
     4081 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
     4141 ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc
     4201 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
     4261 accttaccag agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc
     4321 cagtctagct atcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg
     4381 ttttcccttg tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc
     4441 ggactggctt tctacgtgtt ccgcttcctt gttttagagc tagaaatagc aagttaaaat
     4501 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt
     4561 tttatgtctc ggcgaattgg gtgcggccgc gtttgtca
//
